1. Field of the Invention
The present invention relates to a process for producing a foreign protein in Escherichia coli as a host by recombinant DNA technique.
2. Related Prior Art Statement
Where a foreign protein is produced in E. coli by recombinant DNA technique, some foreign proteins are highly sensitive to proteases in E. coli and their productivity is therefore extremely poor in an ordinary expression system. Even if a foreign protein is not highly sensitive to protease in E. coli, many proteins are somehow affected by proteases so that their yield decreases.
T4 phage is one of the known virulent phages, a host of which is E. coli, and it has an excellent ability of producing, e.g., an inhibitor on host protease(s) which are involved in degradation of abnormal proteins [Simon et al., Nature, 275, 424 (1978)]. For this reason, it is extremely beneficial from an industrial viewpoint to apply various functions of T4 phage, especially the ability to produce an inhibitor on host protease(s) to the production of useful substances by recombinant DNA technique using E. coli as a host.
For the purpose of directly utilizing the functions of T4 phage, research has been heretofore conducted to clone a DNA fragment encoding a foreign protein to the T4 phage genome to convert the T4 phage itself into an expression vector [Casna et al., Gene, 18, 297 (1982), Noguchi et al., Gene, 44, 133 (1986), Mattson et al., Japanese National Publication No. 60-502187]. All of this research utilizes the DNA recombination activity of T4 phage, based on the replacement by DNA recombination in vivo of T4 phage DNA cloned in a plasmid with the genome of T4 phage infected to a host due to their DNA homology, thereby to introduce the DNA fragment encoding a foreign protein into phage genome. According to this technique, a suitable T4 phage promoter ligated with the gene encoding a foreign protein to be expressed can be introduced the phage genome, thus at least succeeding to render the T4 phage itself an expression vector [Casna et al., Gene, 37, 31 (1985), Noguchi et al., Japanese Patent Application Laid-Open No. 62-232384].
T4 phage carries hydroxymethylcytosine (HMC), which is an abnormal base, in its DNA strand instead of normal cytosine (C). When mutations are introduced into genes 42, 56, denB and alc, however, the HMC is completely substituted with normal C [Snyder et al., Proc. Natl. Acad. Sci. (USA), 73, 2098 (1976)]. Such a multiple mutant is generally called T4dC phage. Since T4dC phage is deficient in denB gene(endonuclease IV-deficient), the phage has an extremely high frequency of DNA recombination with a plasmid as compared to a wild type phage. Therefore, in the prior art technique, after the infection of T4dC phage, DNA recombination with a plasmid is caused to construct recombinant T4dC phage [Noguchi et al., Gene, 44, 133 (1986)]. However, T4dC phage is a multiple mutant so that its proliferation ability is poor as compared to a wild type phage and its productivity of an expressed product also decreases. In order to express the desired foreign protein in a high quantity, in the prior art technique it was thus necessary to construct HMC recombinant phage by further hybridization of the recombinant T4dC phage with a wild type phage.
As stated above, it was very complicated to render T4 phage itself an expression vector, including preparation of a hybrid plasmid. Furthermore, an amount of the desired foreign protein to be expressed was not sufficiently satisfactory [Noguchi et al., Japanese Patent Application Laid-Open No. 62-232384, Casna et al., Gene, 37, 31 (1986)].